Selection of appropriate pollination states using phenological and morphological characteristics in five varieties of Phalaenopsis orchid

Introduction

The Orchidaceae is one of the largest families of flowering plants with about 28,000 species classified into around 763 genera (Christenhusz and Byng 2016). The Phalaenopsis is a monopodial orchid in the Orchidaceae family. The genus Phalaenopsis comprising over 60 species belongs to the tribe Vandaeae under subfamily Vandoideae, which contains five subgenera, viz. subgenus Proboscidioides, subgenus Aphyllae, subgenus Parishianae, subgenus Polychilos, and subgenus Phalaenopsis (Christenson 2001).

Darvin (1862) was the first scientist that studied the pollination of orchids and showed that orchids are propagated by insects. Other researchers pointed out the pollination orchid by bees, flies, beetles and birds (Liu et al. 2010). Lack of nutrients in the nectar of orchid flowers is one  of   the  problems  of   natural  pollination  by

pollinators, but variation in color and scent of flowers that attracts insects compensates this problem in the food-deceptive orchids (Li et al. 2012).

Breeding new varieties of Phalaenopsis is time-consuming. New hybrid seedlings are normally derived from crosses of two high-quality parental varieties with different characteristics. In general, breeding programs are designed to improve the size and color of the flowers, as well as other characteristics such as longevity, stalk, length, leaf shape, ease of cultivation, or disease resistance (Tang and Chen 2007).

The crossing of orchids has economic importance due to the variety of colors, different flower size and flower shape (Hartati 2009). The result of crossing between swamp orchid (Phaiussps), one of the endangered species of orchids, and two native species of India, Phaius tankervilleae (Banks ex l’Heritier) Bl and Phaius flavus (Blume) Lindl, showed that crossability between species leads to new hybrids, which have petals and sepals different in color and size from the female parent (Devades et al. 2019). In a research program, compatibility between 23 species and 14 hybrids of Dendrobium was studied (Devades et al. 2016). The pod setting in the species byspecies crosses wasonly 8.97% in the direct crosses and 18.75% inthe reciprocal crosses. On the other hand, the rate of pod formation was 34.37%in the direct species ×hybrids crosses and 50% in theirreciprocal combinations. In another study, seven commercial Phalaenopsis hybrids were cross-pollinated with each other. The cross-fertilization rate was 76.7%.

Also, 19 new hybrids were produced from these crossings (Lesar et al. 2012).

Hicks (2000) stated that a plant with smaller flowers should be used as a female plant and a plant with larger flowers as a male plant. Pollen of smaller flowers, when germinating on the stigma of a larger flower, may not develop a pollen tube long enough to reach the egg cell in the ovarian of the larger flowers.

The purpose of this study was to evaluate five Phalaenopsis varieties under self-pollination conditions and also as the female or male parents in the crosses for six phenological and morphological traits.

Material and Methods

To compare five different Phalaenopsis varieties as male or female parents and the effect of gender on different traits after pollination, a factorial experiment was designed based on a completely randomized design with three replications in a greenhouse of Fardis town in the Alborz province, Iran, from 2018-2019 growing season. The varieties as females were the levels of the first factor, and as males were the levels of the second factor. The Phalaenopsis varieties under study were as follows: Nottingham (W), Bucharest (R), Memphis (P), Dubrovnik (Y) and Andorra (K). The greenhouse had a temperature range of 20-27 ^oC, the humidity of 80% and light of 2500 lx. To pollinate the orchid varieties, an orchid pollination syringe was invented (Patent number: 100033). The syringe consisted of a plastic body, two replaceable toothpicks as pollination needles, a needle adjustment screw, a chassis with a 30^o angle from the syringe body and a light-angle adjustment screw. Plants were pollinated by self and cross-pollination. After the toothpick strikes the male organ in the orchid flower, it is sucked into the inner space of the syringe by the piston, and therefore, the possibility of falling to the ground and becoming contaminated is reduced. Also, the syringe is easily pressed into the female organ (whether in the self-pollination or cross-pollination conditions) and thus pollination is done. The measured characteristics were as follows: time to the first pollination (TFP), when wilting of the petals was visible, time to the first sign of capsule swelling (TCS), capsule length (CL) in centimeters, the weight of the full capsule (WFC) in grams, the weight of the empty capsule (WEC) in grams, the weight of seeds in each capsule (WSC) in grams (the difference between WFC and WEC).

After analysis of variance of the data, the means were compared by Duncan’s multiple range test using the SAS software.

Results and Discussion

Time until the first pollination signs (TPS)

TPS varied in different cultivars. The differences among male parents (p≤ 0.05) and the male by female interaction (p≤ 0.01) were significant on TPS. However, the differences among female parents were not significant. Bucharest as the male parent showed the signs of petal-wilting later than other varieties (6.63 days, which was significantly different from Nottingham as the earliest variety (5.92 days) (Figure 1).

Figure 1.  Time until the first pollinationof the Phalaenopsis varieties as the male parent.

Comparing the TPS of the five self-pollination and 20 cross-pollination states showed that Nottingham had significantly lower TPS (4.6 days) than other states at the self-pollination condition. After that, Andorra × Memphis and Bucharest × Andorra with 5.8 days were better than the rest; however, they were not significantly different from some other combinations. Petals displayed signs of wilting later in Nottingham × Andorra,  Memphis ×  Dubrovnik and Bucharest ×

Table 1. Means for various pollination states (self-pollination and cross-pollination) for the Phalaenopsis characters under investigation.

Means within each trait with the same letter(s) are not significantly different at the 5% probability level according to Duncan’s multiple range test.

Bucharest (Table1).

Time until the first signs of capsule swelling (TCS)

The effect of male and female parents and their interaction on TCS was significant (p≤ 0.01). Nottingham was a better variety either as a female or male parent under self-pollination (16.76 and 15.68 days, respectively) and had significantly lower values than other varieties (Figures 2 and 3).

Table 1 shows the data about TCS values for different self-pollination and cross-pollination states. Self-pollination of Nottingham showed the earliest sign of capsule swelling (13.6 days) and Dubrovnik × Nottingham, Bucharest × Bucharest, Dubrovnik × Memphis, Memphis × Bucharest and Dubrovnik × Andorra equally ranked second in earlier capsule swelling (15.8 days). Nottingham × Andorra had the longest TCS (24.2 days), however, it was not significantly different from the  Bucharest  ×  Dubrovnik.   Self-pollination  of

Figure 2. Time until the first signs of capsule swelling of the Phalaenopsis varieties as the female parent.

Figure 3. Time until the first signs of capsule swelling of the Phalaenopsis varieties as the female parent.

Bucharest and Nottingham (15.8 and 13.6 days, respectively)  caused  the  earlier capsule swelling and consequently, seed setting than their corresponding hybrids Nottingham × Bucharest and Bucharest × Nottingham (22 and 19.2 days, respectively). On the other hand, opposite conditions happened concerning Dubrovnik and Memphis, and self-pollination caused capsules to swell later than their corresponding crosses. Also, the cross of Memphis × Nottingham had higher TCS than Nottingham. Dubrovnik had a TCS value of 19.2 days when self-pollinated, while its value reduced by crossing to Nottingham [Dubrovnik × Nottingham (15.8 days)]. In other words, crossing improved this trait in the Dubrovnik  variety,   but   the   crossing   was   not

beneficial for the Nottingham variety and caused a decline in its TCS.

Weight of full capsule (WFC)

The results of data analysis showed that the effect of female and male parents and their interaction was significant on WFC (p≤ 0.01). According to Figures 4 and 5, Nottingham had the highest WFC both as the female and the male parent (7.51 and 8.54 g, respectively). Memphis had the lowest WFC as the female parent but was not significantly different from the Bucharest and Dubrovnik varieties. The lowest WFC belonged to Dubrovnik (5.5 g) as the male parent (Figure 5).

Figure 4. Weight of the full capsule of the Phalaenopsis varieties as the female parent.

Figure 5. Weight of the full capsule of the Phalaenopsis varieties as the male parent.

Nottingham acquired the highest WFC (9.66 g) under self-pollination conditions as compared to other combinations (Table 1).  After Nottingham, the Dubrovnik × Memphis cross had the highest WFC (8.67). Bucharest × Dubrovnik (5.29 g), Andorra × Memphis (5.31 g) and Nottingham × Bucharest (5.54 g) showed the lowest WFC than other treatments. The crossing of Memphis × Andorra and Andorra × Memphis (6.46 and 5.31 g) decreased WFC as compared to their parents Andorra (7.68 g) and Memphis (7.12 g) under self-pollination conditions. Therefore, larger capsules were obtained when these varieties were self-pollinated. On the other hand, self-pollination of Dubrovnik and Memphis showed lower capsule weight (6.13 and 7.12 g) than the Dubrovnik × Memphis (8.67 g) cross (Table 1) so cross-pollination between these varieties was beneficial as compared to their self-pollination. Also, the cross Dubrovnik × Nottingham and (as the worst and best variety in terms of WFC) showed that cross-pollination improved this trait as compared to the inferior parent (Dubrovnik) and decreased it compared to the superior parent (Nottingham). So cross-pollination with Nottingham (as the male parent) can improve FWC in Dubrovnik (as the male parent). On the other hand, the Andorra × Memphis and Memphis × Andorra crosses showed significantly lower WFC (5.31 and 6.46 g respectively) than their parents [Andorra (7.68 g) and Memphis (7.12 g), respectively]. In other words, cross-pollination between Andorra and Memphis was not suitable concerning this trait (Table 1).

Weight of the empty capsule (WEC)

Effects of different varieties as female and male parents and their interaction on EWC were significant (p≤ 0.01). Based on Figures 6 and 7, Nottingham  had  the  highest  WEC  either  as the

Figure 6. Weight of the empty capsule of the Phalaenopsis varieties as the female parent.

Figure 7. Weight of the empty capsule of the Phalaenopsis varieties as the male parent.

female parent (4.74 g) or as the male parent (5.17 g). Other varieties as female parents were significantly lower than Nottingham for WEC but they were not significantly different from each other. Among the male parents, Dubrovnik showed the lowest WEC (3.56 g).

Nottingham, when self-pollinated, had the highest WEC (6.08) among all treatments Table 1). After Nottingham, Dubrovnik × Memphis (5.27 g), Andorra × Andorra (5.07 g) and Nottingham × Memphis (4.96 g) had higher WEC too, although not significantly different from some other treatments. Nottingham × Bucharest had the lowest WEC (3.47 g). Dubrovnik × Nottingham and Nottingham × Dubrovnik (4.69 and 4.16 g) had significantly lower WEC than the Nottingham parent (6.08 g) and significantly higher WEC than the Dubrovnik parent (3.61 g). In other words, cross-pollination of Dubrovnik with Nottingham improved its WEC either as the female or as the male parent.

The weight of seeds in the capsule (WSC) Effects of the females, males and their interaction were significant on WSC (p≤ 0.01). Nottingham and Andorra as the female parent (2.763 and 2.74 g) had the highest amount of WSC than other varieties but they were not significantly different than Bucharest. As the male parent, Nottingham showed the highest WSC (3.37 g) and Dubrovnik the lowest WSC (1.94 g) among treatments (Figures 8 and 9).

Self-pollination of Nottingham resulted in the highest WSC (3.58) but this value was not significantly different from those of Dubrovnik × Memphis (3.402 g), Nottingham × Memphis (3.34 g) and Dubrovnik × Nottingham (3.24 g). The crossing of Nottingham as the female parent with Dubrovnik as the male parent, which have the highest and lowest WSC, respectively, did not improve this trait over the parents. However, cross-pollination between Dubrovnik and Memphis (i.e. Memphis ×  Dubrovnik  with 3.02 g

Figure 8. The weight of seeds per capsule in the Phalaenopsis varieties as the female parent.

Figure 9. The weight of seeds per capsule in the Phalaenopsis varieties as the male parent.

WSC and Dubrovnik × Memphis with 3.4 g WSC) decreased WSC as compared to Dubrovnik (3.52 g) and increased WSC as compared to Memphis (2.55 g). This means that cross-pollination can be used in the Memphis variety for improving seed yield. Cross-pollination of Nottingham with Andorra was not effective for increasing seed yield over the parents. Although Hicks (2000) indicated that the parent with a bigger flower should be used as the pollen donor but we did not obtain this result about the Nottingham variety that had larger flowers than the other four varieties.

Capsule length (CL)

The result of data analysis showed that the effect

of females, males and their interaction were significant (p≤ 0.01) on WSC. Nottingham either as the female or the male parent showed significantly longer capsules than other varieties. Bucharest, Andorra and Memphis (with 6.719, 6.386 and 5.996 cm CL, respectively) had higher values than Dubrovnik (5.17 cm) as the male parent Figures 9 and 11).

Self-pollination of Nottingham resulted in the highest CL (11.042 cm) among other self-pollination or cross-pollination states. After Nottingham, Dubrovnik × Memphis (8.82 cm), Nottingham × Memphis (8.61 cm), Dubrovnik × Andorra (8.37 cm), Nottingham × Dubrovnik (8.27 cm) and Dubrovnik × Nottingham were significantly better than other treatments. Comparison of CL in Bucharest × Nottingham (5.91 cm) and Nottingham× Bucharest (5.21 cm) showed a decrease in this trait compared with their parents, Nottingham (11.04 cm) and Bucharest (6.36 cm), however, the decrease in Bucharest × Nottingham (5.91 cm) was not significant as compared to Bucharest parent. This means that the capsule length was larger under self-pollination than the cross-pollination in these orchid varieties. On the other hand, Nottingham × Dubrovnik and Dubrovnik × Nottingham (with 8.27 and 8 cm CL, respectively) had higher CL than Dubrovnik (5.31 cm) and lower CL than Nottingham (11.04 cm). CL in self-pollination of Bucharest (6.36 cm) and Andorra (6.08 cm) was lower than the Andorra × Bucharest cross (7.08 cm) and higher than the Bucharest × Andorra (5.65 cm) cross. This means that the cross of Bucharest with Andorra was favored over the self-pollination of both parents when Bucharest was used as the male parent.

According to Hick (2000), if the plant with larger flower used as the female parent and the plant with the smaller flower used as the male parent, the pollen may not grow long enough in the larger flower to  reach  the  ovarian, but in this

Figure 10. The capsule length of the Phalaenopsis varieties as the female parent.

Figure 11. The capsule length of the Phalaenopsis varieties as the male parent.